Abstract

The healthy intestinal mucosa is home to the largest population of macrophages in body. Like all tissue macrophages, intestinal macrophages play vital roles in maintaining tissue homeostasis by removing apoptotic cells and any other cellular debris. In addition they maintain the integrity of the epithelial barrier and support the differentiation and maintenance of regulatory T cells in the mucosa. By virtue of their high phagocytic and bactericidal activity, these macrophages are also vital members of the innate immune system and are strategically positioned adjacent to the epithelium so that they can capture and eliminate any invading organism(s). However unlike other tissue macrophages, those found in the normal gut have several functional adaptations, such as hyporesponsiveness to toll-like receptor (TLR) ligands, which allow them to function without provoking overt inflammation. Macrophages are also abundant during intestinal inflammation, where they show increased TLR responsiveness, pro- inflammatory cytokine and chemokine production and enhanced phagocytic ability. Under these conditions, macrophages perpetuate inflammation. It remains unclear whether these distinct roles in healthy and inflamed intestine roles are carried out by discrete populations of macrophages, or if the resident macrophages alter their behaviour and become pro-inflammatory. One of the main obstacles to gaining a better understanding of the immunobiology of intestinal macrophages during steady state and inflammatory conditions is discriminating them from other mononuclear phagocytes (MP) in the mucosa, such as dendritic cells (DC). At the time of starting my project, it was becoming clear that markers such as F4/80 and CD11c were insufficient for distinguishing between macrophages and DC when used in isolation. Therefore, the aims of this thesis were to first establish reliable multi-parameter flow cytometry staining protocols to allow precise phenotypic and functional characterisation of macrophages in the healthy and inflamed mouse colon, and secondly, to explore the origins of these macrophage populations to assess whether they were derived from distinct precursors, or whether a relationship existed between them. Lastly, I examined the potential mechanisms underlying the characteristic TLR hyporesponsiveness that intestinal macrophages exhibit, focusing on the role of the inhibitory CD200R1-CD200 axis.
In Chapter 3, I first set out to characterise phenotypically the macrophage populations present in the steady state mouse colon using multi-parameter flow cytometry. These studies revealed that expression of the chemokine receptor CX3CR1 could be used to identify two main populations of myeloid cells, the bigger of which was a homogeneous population of CX3CR1highCD11b+ macrophages that dominated the resting mucosa. A smaller population of CD11b+ cells expressing intermediate levels of CX3CR1 (CX3CR1int) was also present in the steady state mucosa, but this was remarkably heterogeneous, with at least 4 subsets distinguishable on the basis of Ly6C, class II MHC, F4/80 and CD11c expression. These included F4/80+Ly6ChighMHCIIneg CD11cneg cells that were phenotypically indistinguishable from blood monocytes, F4/80+Ly6C+MHCII+CD11c+/neg cells and F4/80+Ly6CnegMHCII+CD11c+/int cells that were phenotypically and morphologically similar to CX3CR1high macrophages except for their lower level of CX3CR1. Finally there was a minor subset of F4/80negLy6Cneg MHCII+CD11chigh cells that expanded markedly in response to in vivo flt3L treatment and appeared to be genuine DC. CX3CR1neg cells were also found within the CD11b+ population in the healthy mucosa, most of which were Siglec F+ eosinophils, together with a few neutrophils. In the second half of Chapter 3, I examined how these populations changed during acute colitis induced by feeding dextran sodium sulphate (DSS). These experiments demonstrated that the CX3CR1int compartment expanded dramatically during acute inflammation, with preferential accumulation of the Ly6Chigh subsets and relative loss of the CX3CR1high population as colitis progressed. Together these studies suggested that CX3CR1high and CX3CR1int cells represent resident and pro-inflammatory macrophages respectively.
I next set out to explore the in vivo origin of the CX3CR1int and CX3CR1high populations, to address whether they were derived from independent precursors as would be predicted by current theories of monocyte heterogeneity, or if a relationship existed between them. By using adoptive transfer of purified BM monocytes, the studies described in Chapter 4 show that 'inflammatory' Ly6Chigh, but not Ly6Clow 'resident' monocytes replenished the CX3CR1high resident macrophage population in the steady state mucosa. This appeared to involve local differentiation of Ly6Chigh monocytes through CX3CR1int intermediary stages, which was accompanied by the acquisition of class II MHC, loss of Ly6C and upregulation of F4/80 and CX3CR1. In vivo BrdU incorporation studies supported the idea that the majority of CX3CR1int cells in the resting intestine represented short-lived intermediaries on their way to becoming CX3CR1high macrophages. Together these studies suggested that rather than representing independent macrophage subsets, the CX3CR1int and CX3CR1high cells in the resting colonic mucosa comprise a differentiation continuum from Ly6Chigh monocytes to mature CX3CR1high macrophages. Analysis of BM chimeric mice confirmed that BM-derived monocytes were the source of the vast majority of colonic LP macrophages. These findings were supported by the fact that CCR2 KO mice, in whom Ly6Chigh monocyte egress from the BM is blocked, lack Ly6Chigh colonic monocytes and have markedly reduced numbers of mature colonic macrophages. In Chapter 4, I also explored whether factors present in the normal mucosa, such as colony stimulating factor (CSF)-1, TGFbeta and the chemokine CX3CL1, could direct monocytes to acquire the phenotype of mucosal macrophages. Although initial in vitro studies suggest that none of these were effective on their own, in vivo administration of recombinant CSF-1 appeared to promote in situ monocyte differentiation in the gut. Taken together, the results in this chapter highlight that the CX3CR1high macrophage population is maintained by Ly6Chigh blood monocytes and that their differentiation is controlled by local factors in the mucosa. In Chapter 5, I went on to investigate whether the phenotypically identifiable differentiation of mucosal macrophages was accompanied by alterations in their functional capacity. Intracellular cytokine staining, qRT-PCR and reporter gene expression revealed that as Ly6Chigh monocytes differentiate locally through the CX3CR1int stages into CX3CR1high macrophages, they progressively acquired the ability to produce IL10 and have reduced production of pro-inflammatory mediators. In addition, the maturation of monocytes was accompanied by an increased ability to phagocytose and kill bacteria. Their response to exogenous stimulation by TLR ligands also altered as differentiation proceeded, with the Ly6Chigh monocytes responding robustly to TLR2 and TLR4 ligation in a TNF-alpha dominated manner, whereas the CX3CR1high macrophages responded less vigorously and their TNF-alpha production was balanced by IL10. This pattern was retained during experimental colitis, where the CX3CR1int cells showed enhanced spontaneous TNF-alpha production, whereas IL10 remained the dominant product of CX3CR1high macrophages. Adoptive transfer experiments in Chapter 6 then showed that donor Ly6Chigh monocytes were recruited to the mucosa of colitic mice, but unlike in resting mice, they failed to acquire the CX3CR1high phenotype. These results supported the idea that in inflammation, local differentiation of monocytes is arrested at an early stage before anti- inflammatory macrophages can develop and that this allows inflammation to be perpetuated. In Chapter 6 I also examined the role of inflammatory monocytes in the pathogenesis of colitis, again taking advantage of CCR2 KO mice. These mice showed reduced susceptibility to acute DSS colitis and this was associated with a lack of monocytes and their immediate descendents in the colon of resting KO animals. In parallel, CCR2 KO monocytes were unable to migrate to inflamed colon and together these findings confirmed that Ly6Chigh monocytes are essential for the development of the pathology. Further experiments in Chapter 6 examined how the colonic macrophage populations changed during the resolution phase of colitis. Within two weeks of withdrawing DSS, there was partial restoration of the CX3CR1high macrophage population and this was paralleled by a loss of the CX3CR1int cells that had accumulated during inflammation. However, in vivo BrdU-labelling studies revealed that the reappearance of CX3CR1high macrophages was probably not due to the subsequent differentiation of recruited CX3CR1int cells. These results imply that many of the Ly6Chigh monocytes recruited during inflammation give rise to short- lived effector cells and that restoration of the resident macrophages population may involve de novo recruitment of blood monocytes. Finally, in Chapter 7, I explored some of the mechanisms that might underlie the hyporesponsiveness that intestinal macrophages exhibit. In particular, I examined the role of the CD200R1-CD200 regulatory axis, as this has been shown to control macrophages activity in other tissues. First, I established that resident colonic macrophages express CD200R1 and that its ligand is expressed in the resting colon by both haematopoietic and non-haematopoietic cells. I then went on to examine the impact of CD200R1-deficiency on the colonic macrophages pool by taking advantage of CD200R1 KO mice. This revealed that although these mice had an additional population of F4/80+MHCIIint colonic macrophages, these lacked Ly6C expression and so were unlikely to represent recently recruited pro-inflammatory macrophages. Colonic macrophages from CD200R1 KO mice also expressed comparable levels of costimulatory molecules and TLR to WT mice, and showed no signs of hyper-reactivity to exogenous stimuli. Furthermore, neither CD200R1-deficiency nor lack of CD200 resulted in the development of spontaneous intestinal inflammation, or increased susceptibility to DSS colitis. Final experiments in this chapter suggested that deliberate ligation of CD200R1 on CSF-1 generated BM-derived macrophages had little or no effect on the responsiveness of these cells to innate stimuli. Taken together, the data in Chapter 7 suggested that unlike alveolar macrophages and microglia, the CD200R1- CD200 axis plays little or no role in controlling colonic macrophage activity. Collectively, the results presented in this thesis make important steps forward in our understanding of the complex network of MP in the colon during homeostasis and inflammation. By using precise characterisation of mucosal myeloid cell subsets, I have been able to show that 'inflammatory' Ly6Chigh monocytes constantly enter the steady state mucosa, where they differentiate locally through a series of CX3CR1int intermediaries to replenish the majority CX3CR1high macrophage population. This maturation process is accompanied by alterations in function, so that resident CX3CR1high macrophages are relatively desensitised to exogenous stimuli, but retain high phagocytic activity and produce IL10 constitutively. The exact factors that influence monocyte maturation and control macrophage activity in the normal gut are still unclear, although signalling through CD200R1 alone appears to play no role in this. My studies have also established that pro-inflammatory macrophages in the colon arise from the same Ly6Chigh monocyte precursor and accumulation of these cells during chemically induced colitis is partly due to the arrest in the local differentiation process. The recruitment of Ly6Chigh monocytes and their derivatives in acute inflammation is dependent on CCR2 and is central for pathology, probably due to their readiness to produce pro- inflammatory mediators such as TNF-alpha. To the best of my knowledge, these results demonstrate for the first time that 'resident' and 'pro-inflammatory' macrophages in the colon are not independent cell types, but rather represent alternative, context-dependent differentiation outcomes of the same monocyte precursor. These findings have important implications, as targeting of inflammatory monocyte infiltration has been considered as a potential strategy for the treatment of inflammatory bowel disorder (IBD). However given my observations that these monocytes also replenish the resident macrophage population, the depletion or blockade of these monocytes may have impact on 'resident' macrophage populations and might impair their immunoregulatory roles.